Bioaccumulative and Conchological Assessment of Heavy Metal Transfer

Nica et al. Chemistry Central Journal 2012, 6:55 http://journal.chemistrycentral.com/content/6/1/55

RESEARCH ARTICLE Open Access Bioaccumulative and conchological assessment of heavy metal transfer in a soil-plant-snail food chain Dragos V Nica1, Marian Bura1, Iosif Gergen2, Monica Harmanescu3 and Despina-Maria Bordean2*

Abstract Background: Copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) can pose serious threats to environmental health because they tend to bioaccumulate in terrestrial ecosystems. We investigated under field conditions the transfer of these heavy metals in a soil-plant-snail food chain in Banat area, Romania. The main goal of this paper was to assess the Roman snail (Helix pomatia) usefulness in environmental monitoring as bioindicator of heavy metal accumulation. Eight sampling sites, selected by different history of heavy metal (HM) exposure, were chosen to be sampled for soil, nettle leaves, and newly matured snails. This study also aimed to identify the putative effects of HM accumulation in the environment on phenotypic variability in selected shell features, which included shell height (SH), relative shell height (RSH), and whorl number (WN). Results: Significantly higher amounts of HMs were accumulated in snail hepatopancreas and not in foot. Cu, Zn, and Cd have biomagnified in the snail body, particularly in the hepatopancreas. In contrast, Pb decreased when going up into the food chain. Zn, Cd, and Pb correlated highly with each other at all levels of the investigated food chain. Zn and Pb exhibited an effective soil–plant transfer, whereas in the snail body only foot Cu concentration was correlated with that in soil. There were significant differences among sampling sites for WN, SH, and RSH when compared with reference snails. WN was strongly correlated with Cd and Pb concentrations in nettle leaves but not with Cu and Zn. SH was independent of HM concentrations in soil, snail hepatopancreas, and foot. However, SH correlated negatively with nettle leaves concentrations for each HM except Cu. In contrast, RSH correlated significantly only with Pb concentration in hepatopancreas. Conclusions: The snail hepatopancreas accumulates high amounts of HMs, and therefore, this organ can function as a reliable biomarker for tracking HM bioavailability in soil. Long-term exposure to HMs via contaminated food might influence the variability of shell traits in snail populations. Therefore, our results highlight the Roman snail (Helix pomatia) potential to be used in environmental monitoring studies as bioindicator of HM pollution. Keywords: Helix pomatia, Bioindicator, Environmental monitoring, Heavy metal accumulation, Food chain, Risk assessment

Background monitoring relies on specific analytical techniques (e.g., The growing awareness and concerns about the impact titrimetric methods, atomic spectroscopy, mass spec- of global pollution on human lives and health have tri- trometry, chromatography) to measure the precise level ggered scientists interest to characterize and monitor of specific pollutants in natural environments, but it the quality of the biota. Environmental monitoring is cannot account for the cumulative impact of complex defined as “a time series of measurements of physical, mixtures [2,3]. Biological monitoring uses instead chemical, and/or biological variables, designed to answer selected biological responses (e.g., morphological altera- questions about environmental change” [1]. Chemical tions, cellular biomarkers, behavioral changes, etc.) deriving from environmental exposure to complex mix- * Correspondence: [email protected] tures of chemicals for evaluating exposure and health 2Banat's University of Agricultural Sciences and Veterinary Medicine from risk compared to an appropriate reference [4]. These Timisoara, Faculty of Food Processing Technology, Calea Aradului 119, RO two complementary approaches have been successfully 300645, Timisoara, Romania Full list of author information is available at the end of the article integrated in most environmental monitoring programs

© 2012 Nica et al.; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nica et al. Chemistry Central Journal 2012, 6:55 Page 2 of 15 http://journal.chemistrycentral.com/content/6/1/55

to provide information feedback on the actual impact of included these HMs among the most dangerous sub- human-induced pollution on the environment, and to stances discharged into the natural environment. reflect the efficiency of prescribed mitigation measures The Banat region is a highly industrialized area in to protect the environment [5]. western Romania, which includes the studied areas. Besides the persistent organic pollutants, heavy metal The diverse mineral resources of this region have (HM) accumulation in air, soil, water, and biota is in- facilitated the extensive development of mining, mi- creasingly becoming a global problem with the develop- neral, and metalurgical industry, particularly in the ment of industry, mining activity, application of sewage county of Caras-Severin. Most of these industrial facil- sludge, and irrigation of waste water [2]. HM pollution ities were established under Habsburg administration of the soil is often less visible and direct than other types in the eighteenth century and continued till the end of of land pollution (e.g., garbage, refuse, littering), but its the tweentieth century (e.g., Resita, Otelu Rosu, Anina, effects on terrestrial ecosystems and humans are long- Bocsa, etc.) [17]. Long-term pollution in the county of lasting and severe [5]. HM toxicity in the environment Caras-Severin resulted in Cu, Zn, Cd, and Pb accumu- depends on several physicochemical and biological fac- lation in soil and vegetation [18]. Another major in- tors, among which HM bioavailability in soil plays a cru- dustrial area in Banat region is the town of Timisoara, cial role in their accumulation along the food chain; the largest and the most populated city from this area. therefore, this factor becomes an important issue in Recent studies occasionally revealed significant Cu, Zn, assessing the environmental quality [6]. It was also Cd, and Pb levels in air, soil, water, and vegetation in found that synergic and antagonic relationships existing Timisoara and the neighbouring areas [19]. However, between HM metal ions may increase their toxic poten- little information is available concerning the bio- tial [7,8]. Such findings are extremely important, because accumulation and the biomagnification of these HMs in the environment HMs usually co-occur in complex in terrestrial ecosystems in Romania, particularly in mixtures [9]. Banat region. Copper (Cu), zinc (Zn), cadmium (Cd), and lead Many studies [20-23] showed that land snails can con- (Pb) were chosen because they are among the most centrate high levels of Cu, Zn, Cd, and Pb in their soft common HMs which can pollute the environment, tissue without revealing any major metabolic disorders. especially in areas with high anthropogenic pressure. These HMs were found to accumulate into the snail People have been exposed to these HMs on large shell, often resulting in alterations of shell geometry or scale over the last century, and all of them are size [20,24-26]. In polluted soils Cu, Zn, Cd, and Pb are known to pose serious threats to terrestrial ecosys- taken by vegetation, and subsequently they can be trans- tems [5]. The Romanian Soil Quality regulations set fered along food chain to herbivorous organisms. In up the alert threshold level (ATV) to which these addition, Cu, Zn, Cd, and Pb accumulate in dead plant HMs accumulate in soil: Cu: 100 mg kg-1 d.w.; Zn: and animal material, and therefore, they are ingested by 300 mg kg-1 d.w.; Cd: 3 mg kg-1 d.w.; Pb: 50 mg kg-1 organisms that feed on decaying organic matter (i.e., de- d.w. – the values are expressed as miligram per kilo- tritivore organisms). Because snails act on trophic level, gram dry weight (mg kg-1 d.w.) [10]. Cu and Zn have both as herbivorous and detritivore organisms, they can essential physiological and biochemical functions in be involved in these HM biomagnification along the plants and animals [11,12], but excessive levels can food chain [22,27]. Overall, terrestrial gastropods have be damaging to environmental health [3]. For ex- the potential to be used in environmental monitoring as ample, Cu(II) ions (> 40 mg kg-1 d.w.) significanly in- model invertebrate for Cu, Zn, Cd, and Pb accumulation hibit the alfalafa (Medicago sativa L.) seed capacity in terrestrial ecosystems [28]. to germinate and grow [13] whereas exposure to high The present research investigates land snails as end- Zn levels in soil (> 500 mg kg-1 d.w.) reduce the points of Cu, Zn, Cd, and Pb transfer in a soil-plant- ability of plants to absorb iron (Fe) and manganese herbivore food chain in terrestrial ecosystems of the Banat (Mn) [14]. In contrast, Cd and Pb have no known region. It compares the accumulation of these HMs in vital or beneficial effect on organisms [15], except for eight populations of snails from habitats exposed to indus- diatoms where a Cd-based enzyme plays an essential trial pollution for more than 30 years. By recording Cu, Zn, role in regulating atmospheric carbon [16]. Pb and Cd, and Pb level in soil, plant, and snail for each popula- Cd are toxic, persistent, and non-biodegradable in tion, HM biomagnification along food chain is traced to as- the environment, and hence, they can be easy bioac- sess the potential of native gastropods to be used in cumulated and biomagnified along terrestrial food environmental monitoring studies. In addition, we have chains [5]. Because all these HMs pose a serious examined the influence of long-term of HM exposure on toxic threat to human health and ecosystem integrity, intraspecific variation in shell morphology(shell size, rela- the EU Council Directive 76/464/EEC of 4 May 1976 tive shell height, whorl number). Nica et al. Chemistry Central Journal 2012, 6:55 Page 3 of 15 http://journal.chemistrycentral.com/content/6/1/55

Results and discussions different level and history of HM exposure. Both Copsa HM accumulation and transfer in soil–nettle-snail food chain Mica and Biesboch areas are well known in Europe as ‘hot- HM accumulation in soil spots’ of HM pollution [32,33] whereas the Banat area may Our results showed that Cu, Zn, Cd, and Pb concen- be regarded as a low to moderate HM polluted area [34]. trations in soil were significantly different among sam- HM concentrations within the soil compartment, as pling sites (Cu, Zn, Cd: p < 0.01; Pb: p < 0.05). The shown in Table 1, correlated highly with each other, lowest HM values were registered for the site THR (as except for Cu (r = 0.81-0.94, 0.05 < p < 0.001). Similar shown in Figures 1, 2, 3, 4), and therefore, they vali- metal-metal interactions in soil (r = 0.91-0.95, p < 0.001) dated the choosing of this location as the reference have also been reported in the Biesboch area between site. The maximal Cu and Zn loadings in soil were Cu, Zn, Cd, and Pb [22]. The normal content (NC) found at site THM2 (Figures 1, 2). Soil Cd concentra- and the alert threshold level (ATV) for each investi- tion exhibited the highest value at site THM5 (Figure 3), gated HM were used to assess the risks posed by soil whereas the maximal amount of Pb in soil was detected at HM contamination on environmental health. The site THM4 (Figure 4). Soil HM concentrations were com- values were compiled from the Romanian Soil Quality parable with results of previous pedological studies in regulations[10]. It was found that soil Cu and Zn con- Banat area [18,19,29,30]. However, measured values were centrations were generally within NC values at all sites generally lower than in other areas exposed to intense an- (Figures 1, 2). Although in several cases the soil Cd thropogenic pressure and pollution. Therefore, similar concentrations at sampling sites did exceed NC levels studies that investigated HM food chain transfer to land (THM1, THM5, THM6), the measured values did not snails in Copsa Mica region, Romania [31] and the Bies- reach ATV level (Figure 3). It was found that only Pb boch flood-plain area, the Netherlands [22] frequently content slightly did exceed ATV value at site THM4 found 5-10 times higher levels of HM accumulation in (Figure 4). Hence, in accordance with Romanian Soil background soils. These differences were explained by the Quality regulations [10] it was concluded that all snails

Figure 1 Cu concentration in environmental matrices (soil, nettle leaves, snail body). Legend. SR – HM concentrations in soil at reference site (THR), UR – HM concentrations in nettle leaves at reference site (THR), HPR – HM concentrations in snail hepatopancreas at reference site (THR), SFR – HM concentrations in snail foot at reference site (THR), S1-S7 – HM concentrations in soil at sites 1-7 (THM1-THM7), U1-U7 – HM concentrations in nettle leaves at sites 1-7 (THM1-THM7), HP1-HP7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), SF1- SF7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), ATV – alert threshold level (HM concentration in soil). NC – normal content level (HM concentration in soil). Nica et al. Chemistry Central Journal 2012, 6:55 Page 4 of 15 http://journal.chemistrycentral.com/content/6/1/55

Figure 2 Zn concentration in environmental matrices (soil, nettle leaves, snail body). Legend. SR – HM concentrations in soil at reference site (THR), UR – HM concentrations in nettle leaves at reference site (THR), HPR – HM concentrations in snail hepatopancreas at reference site (THR), SFR – HM concentrations in snail foot at reference site (THR), S1-S7 – HM concentrations in soil at sites 1-7 (THM1-THM7), U1-U7 – HM concentrations in nettle leaves at sites 1-7 (THM1-THM7), HP1-HP7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), SF1- SF7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), ATV – alert threshold level (HM concentration in soil). NC – normal content level (HM concentration in soil). originated in habitats with low HM accumulation in Our results (Table 1) showed that HM interactions soil. However, heightened Cd levels at sites THM1, within the leaf compartment followed the same pattern THM5 and THM6 (Figure 3) are expected to raise ser- as that found in soil, i.e., strong correlations among Zn, ious environmental concerns if their level in the near Cd, and Pb level (r = 0.84-0.90, 0.05 < p ≤ 0.001). In con- future reaches ATV value. trast, other authors [22] have found that only correlations including Cd were significant at the same level HM accumulation in nettle leaves (r = 0.33-0.43, 0.05 < p < 0.001). As shown in Table 1, we Our investigations revealed significant differences in identified an effective soil-plant transfer of Zn and Pb in HM accumulation in nettle leaves at sampling sites the selected food chain (r = 0.76-0.93, 0.05 < p ≤ 0.001). In (Cu,Zn,Cd,Pb:p<0.01).ThehighestCuconcentra- Biesboch area, however, only Zn transfer was reported tion in Urtica dioica leaves was detected at site from the soil to the leaf compartment (Zn: r = 0.45, THM2 (Figure 1). Elevated levels of Zn were found p < 0.001; Cu, Cd, Pb: p > 0.05). Not only the pollution in nettle leaves at sites THM2, THM3, and THM4 level itself can be held accountable for such diverse and (Figure 2), whereas Cd reached the maximal values complex data, but also HM bioavailability in soil is es- at sites THM1, THM2, and THM4 (Figure 3). How- sential for HM soil-plant transfer. The dynamics of this ever, the nettle leaves sampled at site THM3 accu- process is mainly regulated by soil physico-chemical mulated, by far, the highest Pb amount (Figure 4). properties (e.g., total metal content, humidity, clay and Interestingly, HM loadings in nettle leaves compared hydrous oxide content, organic matter, pH, redox condi- fairly well with most reported values in Biesboch area tions) [35]. Overall, it was suggested that the pattern of [22], but they were much lower than in Copsa Mica HM accumulation and transfer in nettle leaves resulted area, particularly for Cd, Zn, and Pb [31]. Therefore, from the cumulative action of low soil Cd bioavailability pollution intensity can be an important fostering fac- to plants [9,19] and significant HM content of soils in tor in HM transfer in terrestrial ecosystems. Banat area [36]. Nica et al. Chemistry Central Journal 2012, 6:55 Page 5 of 15 http://journal.chemistrycentral.com/content/6/1/55

Figure 3 Cd concentration in environmental matrices (soil, nettle leaves, snail body). Legend. SR – HM concentrations in soil at reference site (THR), UR – HM concentrations in nettle leaves at reference site (THR), HPR – HM concentrations in snail hepatopancreas at reference site (THR), SFR – HM concentrations in snail foot at reference site (THR), S1-S7 – HM concentrations in soil at sites 1-7 (THM1-THM7), U1-U7 – HM concentrations in nettle leaves at sites 1-7 (THM1-THM7), HP1-HP7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), SF1- SF7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), ATV – alert threshold level (HM concentration in soil). NC – normal content level (HM concentration in soil).

HM accumulation in snail body maximal relative difference (%) between the hepatopan- The location had a significant influence on HM burden creas and the foot at site TM1, where the Cd level was in the snail hepatopancreas (Cu, Zn, Cd, Pb: p < 0.01) more than 10 times higher in the snail hepatopancreas and foot (Cu, Zn, Cd, Pb: p < 0.01). Moreover, highly sig- (Figures 1, 4). nificant differences were found when comparing accu- The present study attested to Roman snail the ability mulation of each HM in snail hepatopancreas with that to concentrate trace metals in its tissues, and therefore, in foot (Cu, Zn, Cd, Pb: p < 0.001). Therefore, one can this species was included among the so-called ‘bioindica- conclude that HM accumulation in the snail body tors of accumulation’ [37]. The accumulation pattern depends on both the area of origin and investigated body over the different tissue parts (i.e., hepatopancreas vs. parts (Figures 1, 2, 3, 4). Globally speaking, the snail foot) was consistent with other studies that revealed the hepatopancreas concentrated the highest HM amounts prefferential deposition of most HMs in the Helix at sites THM1, THM2, THM5, and THM6, except for pomatia hepatopancreas [31,38,39]. When compared the Cd whose level was also reported to be elevated at site results with those of other field studies, the measured TM7 (Figures 1, 2, 3, 4). Similar trends of HM accumu- values were generally much lower than in areas with ele- lation were found in the snail foot for Cu and Zn, vated levels and/or exposed to intensive long-term HM whereas Cd and Pb levels were much more pollution [31,38]. homogenous, regardless of locations (Figures 1, 2, 3, 4). Our findings showed that, excepting Pb burden in Although the measured values were slightly higher in hepatopancreas, Cu concentrations in snail foot and hepatopancreas, the Cu and Pb levels in the snail body hepatopancreas were indepedent variables (Table 1). usually varied within the same range (Figures 1, 4). In Interestingly, like in soil and leaf compartment, Zn, Cd, contrast, Zn and Cd burdens in snail hepatopancreas and Pb concentrations correlated highly with each other were always at least 2.0 times higher than in the snail in both hepatopancreas and foot (r = 0.74-0.90, foot (Figures 2, 3). HM concentration registered the 0.05 < p < 0.001). It was therefore assumed that these Nica et al. Chemistry Central Journal 2012, 6:55 Page 6 of 15 http://journal.chemistrycentral.com/content/6/1/55

Figure 4 Pb concentration in environmental matrices (soil, nettle leaves, snail body). Legend. SR – HM concentrations in soil at reference site (THR), UR – HM concentrations in nettle leaves at reference site (THR), HPR – HM concentrations in snail hepatopancreas at reference site (THR), SFR – HM concentrations in snail foot at reference site (THR), S1-S7 – HM concentrations in soil at sites 1-7 (THM1-THM7), U1-U7 – HM concentrations in nettle leaves at sites 1-7 (THM1-THM7), HP1-HP7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), SF1- SF7 – HM concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7), ATV – alert threshold level (HM concentration in soil). NC – normal content level (HM concentration in soil).

HMs have been transferred together in the investigated 0.05 < p < 0.001) [21]. In addition to the exposure to food chains. These results may have resulted from simi- HMs via food uptake, land snails regularly eat soil and lar geogenic sources (e.g., composition of parent rock, they are also exposed to HMs via epithelial contact [40]. soil physico-chemical properties), anthropogenic causes As a result, future studies should be designed to verify (e.g., history of pollution), and/or environmental condi- our hypothesis and to elucidate such complex interactions (e.g., climate, habitat, trophic chain). We hypothe- tions between HMs during their transfer in the biota. sized that such patterns of HM accumulation and Cu-Cu, Zn-Zn, and Cd-Cd correlations between the transfer in the biota may be used as biochemical finger- snail hepatopancreas and foot were highly significant print for tracing and characterizing the environmental (r = 0.73–0.84, 0.05 < p < 0.01), whereas no relationship pollution risks in a specific area. was found for the Pb content (Table 1). Moderate In contrast to our study, low and moderate correla- Cu-Cu correlations were found between the snail foot tions were found among Cu, Zn, Cd, and Pb concentra- and soil (Table 1), highlighting the importance of this tions in Cepaea nemoralis sampled from the Biesboch organ in snail metabolism as an important place of Cu area (r = 0.37-0.64, 0.05 < p < 0.001), except for Cu-Pb storage [23,41]. interactions (p > 0.05). In this case, HM accumulation Studying HM transfer in a soil-nettle-snail food chain was measured for the whole snail body, excluding the in the Biesboch area, moderate correlations (r = 0.41 – shell [22]. HM pairs Cd-Zn and Cd-Pb correlated mod- 0.64, 0.05 < p < 0.001) were found for all HMs in adult erately with each other in Helix aspersa hepatopancreas Cepaea nemoralis (whole body, excluding shell), except (r = 0.34-0.54, 0.01 < p < 0.001). Besides these two HM for Cu-Cu interactions between snail and soil (p > 0.05) pairs, there were also significant correlations between [22]. Helix aspersa was investigated as sentinel organism the whole body Pb and Zn concentrations (r = 0.59-0.65, to exposure to both physiological and xenobiotic HMs p < 0.001) as well as between Cu-Zn and Pb-Zn pairs for (i.e., Cu, Zn: physiological HMs; Cd, Pb: xenobiotic the whole body without hepatopancreas (r = 0.20-0.73, HMs) in a food chain soil-dandelion-snail. There were Nica http://journal.chemistrycentral.com/content/6/1/55 ta.CeityCnrlJournal Central Chemistry al. et

Table 1 Correlations between Cu, Zn, Cd, and Pb concentrations in soil, nettle leaves, and snail body Log Log Log Log Log Log Log Log Log Log Log Log Log Log Log Log

Cu(S) Zn(S) Cd(S) Pb(S) Cu(U) Zn(U) Cd(U) Pb(U) Cu(SF) Zn(SF) Cd(SF) Pb(SF) Cu(HP) Zn(HP) Cd(HP) Pb(HP) 2012, Log Cu(S) 0.95*** 0.31 ns 0.81* 0.67 ns 0.85** 0.83* 0.61 ns 0.45 ns 0.34 ns 0.34 ns 0.26 ns 0.20 ns 0.05 ns 0.25 ns 0.37 ns 6 Log Zn(S) 0.22 ns 0.88** 0.45 ns 0.93** 0.80* 0.69 ns 0.25 ns 0.11 ns 0.11 ns 0.03 ns -0.02 ns -0.11 ns 0.08 ns 0.15 ns :55 Log Cd(S) 0.25 ns 0.47 ns 0.11 ns 0.18 ns 0.31 ns 0.59 ns 0.58 ns 0.58 ns -0.07 ns 0.87** 0.52 ns 0.64 ns 0.81* Log Pb(S) 0.52 ns 0.90** 0.82* 0.76* 0.38 ns -0.02 ns -0.02 ns -0.23 ns 0.029 ns -0.01 ns 0.02 ns 0.10 ns Log Cu(U) 0.46 ns 0.66 ns 0.40 ns 0.90** 0.67 ns 0.50 ns 0.46 ns 0.62 ns 0.60 ns 0.58 ns 0.75* Log Zn(U) 0.90** 0.87** 0.35 ns -0.07 ns -0.07 ns -0.13 ns -0.12 ns -0.07 ns -0.03 ns 0.10 ns Log Cd(U) 0.84* 0.52 ns 0.06 ns 0.08 ns 0.04 ns 0.03 ns 0.14 ns 0.14 ns 0.31 ns Log Pb(U) 0.48 ns -0.16 ns -0.06 ns -0.38 ns 0.05 ns 0.07 ns -0.01 ns 0.27 ns Log Cu(SF) 0.59 ns 0.49 ns 0.24 ns 0.74* 0.67 ns 0.55 ns 0.84** Log Zn(SF) 0.88** 0.74* 0.73* 0.74* 0.90** 0.80* Log Cd(SF) 0.75* 0.59 ns 0.66 ns 0.83* 0.80* Log Pb(SF) 0.21 ns 0.41 ns 0.56 ns 0.39 ns Log Cu(HP) 0.57 ns 0.63 ns 0.89** Log Zn(HP) 0.90** 0.74* Log Cd(HP) 0.80* Log Pb(HP) Note. *** – p < 0.001, ** – p < 0.01, * – p < 0.05, ns – p > 0.05. Abbreviations. Cu(S) – copper concentrations in soil, Cu(U) – copper concentrations in nettle leaves, Cu(SF) – copper concentrations in snail foot, Cu(HP) – copper concentrations in snail hepatopancreas, Zn(S) – zinc concentrations in soil, Zn(U) – zinc concentrations in nettle leaves, Zn(SF) – zinc concentrations in snail foot, Zn(HP) – zinc concentrations in snail hepatopancreas, Cd(S) – cadmium concentrations in soil, Cd(U) – cadmium concentrations in nettle leaves, Cd(SF) – cadmium concentrations in snail foot, Cd(HP) – cadmium concentrations in snail hepatopancreas, Pb(S) – lead concentrations in soil, Pb(U) – lead concentrations in nettle leaves, Pb(SF) – lead concentrations in snail foot, Pb(HP) – lead concentrations in snail hepatopancreas. ae7o 15 of 7 Page Nica et al. Chemistry Central Journal 2012, 6:55 Page 8 of 15 http://journal.chemistrycentral.com/content/6/1/55

found moderate correlations between Zn and Cd concen- on the investigated body parts. Our study showed that trations in the snail body (whole body, whole body without the snail hepatopancreas accumulates significant hepatopancreas, hepatopancreas) and soil (r = 0.52-0.69, amounts of HMs, pointing to this organ importance as p < 0.001). In the case of Pb, highly significant correlations biomarker of HM pollution. were detected only when soil concentrations were com- The dendrogram (Figure 5) shows the results obtained pared to hepatopancreas and whole body Pb content from using hierarchical cluster analysis and squared Eu- (r = 0.26 – 0.40, p < 0.001). In contrast, no significant rela- clidean distance as a criterion of similarity. Therefore, tionship (r = 0.02- 0.08, p > 0.05) was found between soil based on HM loading in snail hepatopancreas, the investi- Cu concentrations and either the whole snail body or snail gated locations can be classified into three main groups body parts [21]. Such discrepant results were attributed to (r = 0.81). The first group corresponds to the most polluted different selectivity in choice of food exhibited by these areas from the investigated sites (THM1 and THM6). Both species of land snails and by different bioavailability of areas are well known for long and intensive exposure to HMs in investigated food chains. HMs. This group is also characterized by the biggest Eu- clidean distance to the other groups. The second group HM biomagnification along food chain soil-nettle-snail corresponds to the cleanest areas in terms of HMs accu- HM accumulation and transfer from soil to nettle have mulated in the snail hepatopancreas. The three sites in this systematically revealed no metal biomagnification at the group are either located in low-anthropized areas (THR) first trophic level, the primary producers (Urtica dioica; or in areas exposed to other types of industrial pollution, Urticaceae) (Figures 1, 2, 3, 4). In contrast, Cu, Zn, and particularly to chemical industry (THM3, THM4). The last Cd concentrations at the second trophic level, the pri- group includes locations placed near potential sources of mary consumers (Helix pomatia; Helicidae), have usually air pollution with HMs: THM2 – power plant, THM5 – exceeded those found at the first trophic level (Figures 1, vehicular traffic, THM7 – steel industry. Therefore, our 2, 3, 4). Interestingly, except for sites THM5, THM7, results suggested that regions with similar HM exposure and THR, the hepatopancreatic and the foot Pb levels can be appropriately grouped by applying cluster analysis were lower than in the nettle leaves (Figure 4). to HM accumulation in snail hepatopancreas. Because land snails can accumulate higher levels of Cu and Cd than the environmental concentrations, they are HM influence on shell architecture generally recognized as “macroconcentrator” species for Estimation of shell size from linear shell dimensions these HMs. In contrast, terrestrial gastropods are The internal volume of a snail shell signifies the amount “microconcentrator” species for Zn and Pb [37]. Our of housing space available for body growth and, thus, results evidenced that Cu, Zn, and Cd have biomagnified along in snail body, particularly in the snail hepatopancreas, whereas Pb tended to decrease when going up into the food chain. This was explained by the fact that soils from Banat area are naturally rich in Zn [36]. Similar findings (i.e., Cu, Zn, and Cd biomagnification and Pb biodepletion in snail body relative to vegetation concentrations) were reported in the Biesboch area for Cepaea nemoralis [22] and in the Copsa Mica area for Helix pomatia [31]. Both studies investigated HM accumulation in a soil-nettle-snail food chain. Evaluating the use of H. aspersa as sentinel for mapping pollution, it was found that mean vegetation (Taraxacum sp.) levels of Cu, Zn, Cd, and Pb were systematically lower than in snail body [21]. Similarly, Zn, Cd, and Pb biomagnified in H. aspersa body via autochthonous plants (Gramina- ceae) gathered at the vicinity of a highway [27]. Such examples illustrate the importance of identifying the key particularities of each food chain (e.g., soil physico- chemical properties, HMs bioavailability, diet compos- Figure 5 Dendrogram of the cluster analysis based on HM ition, climatic conditions) before employing the Roman accumulation in the snail hepatopancreas. HP1-HP7 – HM snail (Helix pomatia) as bioindicator in environmental concentrations in snail hepatopancreas at sites 1-7 (THM1-THM7); – monitoring studies in this area. Nevertheless, the sensi- HPR HM concentrations in snail hepatopancreas at reference site (THR). tivity of Helix pomatia as bioindicator organism depends Nica et al. Chemistry Central Journal 2012, 6:55 Page 9 of 15 http://journal.chemistrycentral.com/content/6/1/55

this feature is directly associated with the snail size [42]. It was found that the level of anthropic pollution is Two conchological parameters, the shell height (SH) and often associated with the shell size. Thus, smaller shells the shell width (SW), are primarily used to estimate the were reported for snails inhabiting more polluted habi- shell volume (SV) from linear shell dimensions [43,44]; tats [44,45,46]. Cepaea nemoralis displayed a negative such formulas are species-specific, but little information correlation (r = -0.61) between the shell volume and the exists regarding the mathematical relationships for cal- Zn concentration in soil [26]. In our study SH correlated culating Helix pomatia SV. highly with HM concentrations in nettle leaves, except Measured values for SH and SW were normally dis- for Cu (Zn: r = - 0.81, p < 0.05; Cd: r = - 0.80, p < 0.05; Pb: tributed (Shapiro-Wilcoxon test, p > 0.05). Although r = - 0.89, p < 0.01; Cu: p = 0.687). In contrast, no both conchological features correlated significantly with significant relationships were found between SH and SV, there was a stronger positive relationship between HM concentrations in soil, hepatopancreas, and foot SV and SH (r = 0.80, p < 0.001) than between SV and SW (0.080 < p < 0.892). (r = 0.63, p < 0.01). It was therefore concluded that for When compared with the reference site (THR), RSH the investigated snail populations SH is a more accurate was found to be significantly higher only at site THM3, predictor of shell size than SW. A similar approach has whereas no significant differences were determined for been already used for assessing shell growth to juvenile the other snail populations (Table 2). The relative shell Helix aspersa exposed to Pb-enriched food [25]. height (RSH) of a snail species relates to the angle of the substrate on which activity occurs [47]. Flatter shells fa- Influence of HM accumulation shell traits cilitate movement on substrates with foliated structures Our results confirmed that all conchological features (e.g., leaf litter), whereas higher shells are more advanta- (SH, RSH, WN) were normally distributed (all p`s > 0.05) geous on substrate with a fine particle structure (e.g., and satisfied the criterion of homoscedasticity (all soil) [48]. Several studies demonstrated that this feature p`s > 0.05). The area of origin had a significant influence is primarily genetically determined, but, within a given on shell traits variation among the eight snail populations form, it may also be influenced by phenotype [49]. (p < 0.001). Post hoc analysis revealed that THR snails Therefore, the sampled snails have originated from habi- had significanly larger shells than the snail populations tats with different substrates or they belong to different inhabiting locations THM1, THM2, THM3, and THM4. subspecies. In contrast, no significant differences were found for Interestingly, RSH correlated highly with Pb concen- shell size between THR snails and either THM5 or trations in the snail hepatopancreas (r = 0.77, p < 0.05), THM7 snails (Table 2). although it systematically revealed no significant

Table 2 The mean values of conchological features from the eight snail populations Sampling site Shell height (SH) Relative shell height (RSH) Whorl number (WN) THM1 Timisoara city Landfill 3.63 ± 0.19*** 0.98 ± 0.03 ns 4.92 ± 0.04*** (Parta-Sag/Timis county) THM2 South Industrial Platform 3.86 ± 0.11** 0.96 ± 0.02 ns 4.94 ± 0.13*** (Timisoara/Timis county) THM3 North Industrial Platform 3.73 ± 0.24*** 1.01 ± 0.04* 4.44 ± 0.15 ns (Timisoara/Timis county) THM4 East Industrial Platform 3.65 ± 0.20*** 0.95 ± 0.02 ns 4.31 ± 0.15* (Timisoara/Timis county) THM5 Timisoara, Communal Road 64 (DC64) 4.26 ± 0.20 ns 0.96 ± 0.04 ns 4.49 ± 0.14 ns (Timisoara/Timis county) THM6 National Road Resita-Caransebes (DN58) 3.90 ± 0.17* 0.97 ± 0.03 ns 4.80 ± 0.09*** (Caras-Severin county) THM7 Principal Street 3.99 ± 0.19 ns 0.95 ± 0.04 ns 4.66 ± 0.16** (Otelu Rosu/Caras-Severin county) THR Salbagelu Nou 4.18 ± 0.28 0.97 ± 0.03 4.47 ± 0.14 (Caras-Severin county) Note. *** – p < 0.001, ** – p < 0.01, * – p < 0.05, ns – p > 0.05 (Anova, posthoc Dunnett’s test). Figures in bold type defines values significantly lower than the control group (THR), whereas the bold and underlined values are significantly higher than the control group (THR). Nica et al. Chemistry Central Journal 2012, 6:55 Page 10 of 15 http://journal.chemistrycentral.com/content/6/1/55

relationships with HMs content along the soil-nettle- be attributable to close association in background soils snail food chain (0.054 < p < 0.487). Long history of Pb- of the eight sites examined here. Therefore, it is sug- exposure was associated with more robust shells as gested that HM accumulation in investigated areas imply related to width/height ratio [24]. The aforementioned a specific biochemical fingerprint. However, HMs in correlations showed that there are no significant Pb-Pb hepatopancreas appear to be independent of concentra- interactions between snail foot and hepatopancreas. It tion in soil and nettle leaves, except for Cu levels in soil was therefore suggested that investigated snail popula- and snail foot. In the light of present data, the environ- tions may have adopted different strategies to deal with mental monitoring with snails must be extended to HM exposure to Pb. Such strategies have been already mixtures since such associations are regularly found in described in the case of Helix aspersa for which Pb bio- soil. availability in different populations was reversely related Long-term Cd and Pb uptake via vegetation ingestion with soft tissue growth rates and food Mg content [50]. is assumed to be associated with smaller shells and The area of origin exhibited a significant bidirectional higher whorl numbers. Because land snails often exhibit influence on WN (Table 2). Thus, THM1, THM2, intra-annual cycles of activity interspersed by periods of THM6, and THM7 had significantly more whorls than dormancy, this perspective must be more detailed inves- THR snails (p < 0.05), whereas only THM4 snails tigated to establish the real impact of anthopic HM pol- revealed significantly less whorls (p < 0.05). HM levels in lution on wild snail populations. soil, snail hepatopancreas and foot were not correlated with WN (0.054 < p < 0.922), except for soil Cu content Experimental which was highly correlated with WN (r = 0.72, p < 0.05). Study animals and collecting sites Metabolic needs (i.e., Cu-based respiratory hemocya- The Roman snail (Helix pomatia Linnaeus, 1758) is a nins) may explain our results as Cu occurs in the envir- common East and Central European snail species of the onment at concentrations near the minimum nutritional family Helicidae. In Romania, it inhabits forests, gardens, requirements of invertebrates [51]. According to our vineyards, and open habitats that are confined to calcar- results, WN was strongly positively correlated with both eous substrate. The systematic classification was deter- Cd and Pb levels in nettle leaves (Cd: r = 0.84, p < 0.01; mined according to morphometric criteria [54]. To Pb: r = 0.81, p < 0.01), but displayed no significant rela- provide representative and homogenous data, we tionships with Cu and Zn concentrations in the leaf designed our experiments to include only newly matured compartment (Cu: p = 0.058; Zn = 0.059), which has not specimens of Roman snail (Helix pomatia). The snails been reported yet. were collected, during May 2011, from eight locations in When the apertural rim folds back and forms the aper- the Banat area, Romania (Timis and Caras-Severin coun- tural lip the shell growth ends; thus, from then on WN ties), as shown in Figure 6. Being located in a low- remains constant throughout the snail life. Calcium car- anthropized area, away from major sources of pollution bonate deposition is carried out at a relatively constant rate [55], the vilage of Salbagelu Nou (Caras-Severin county) [52]. In addition, within the same populations snails of has been chosen as reference site (THR). All the other similar sizes have equal whorl number [53]. WN can there- sampling sites shared one set of environmental condi- fore be regarded as a rough estimator of snail maturation tions deemed important for assessing the reliability of age. As a result, it was suggested that long-term exposure Roman snail as bioindicator species in the study unit. to Cd and Pb contaminated food may affect snail popula- Therefore, all locations have been exposed for at least tions by decreasing snail size and delaying the maturation 30 years to industrial pollution, and were located within age because of increased detoxification effort. a 10 km-radius of former and/or actual major sources of contamination (see Table 3). At each plot at least 60 spe- Conclusions cimens were sampled from a few square meters, and Given the significant ability to concentrate HMs in its split into three equal samples. To distinguish the newly body, the Roman snail (Helix pomatia) is a relevant ac- matured snails from their elder counterparts we used cumulation bioindicator of long-term metallic exposure the aperture lip as a benchmark of maturation. Older under field conditions. The snail hepatopancreas proved snails had a hardened, thickened, and turned-out aper- to be extremely sensitive to HM traces from soil and ture lip. In contrast, the newly matured juveniles possess have frequently accumulated several times higher a thinner and softer aperture lip. amounts of Cu, Zn, and Cd than in lower trophic levels (soil and nettle); therefore, this organ can be used for Preparation of snails and conchological measurements tracking HM bioavailability in soil. The snails were placed in clean glass containers and Zn, Cd, and Pb appear to interfere with each other in fasted for 48 h (the faeces were removed after 24 h). primary producers and consumers, although this might They were sacrificed by freezing (at −20°C) and stored Nica et al. Chemistry Central Journal 2012, 6:55 Page 11 of 15 http://journal.chemistrycentral.com/content/6/1/55

Figure 6 Map showing the locations of soil, nettle leaves, and snail sampling sites. Legend. 1 – site THM1; 2 – site THM2; 3 – site THM3; 4 – site THM4; 5 – site THM5; 6 – site THM6; 7 – site THM7; R – site THR. until processing in the lab (chest freezer). After defros- albumen gland) and the foot were studied in this work ting, the whole soft body was removed from the shell because these organs are most often reported as the and the viscera and the foot were separated. Only the main sites of metal accumulation and storage in snails hepatopancreas (excluding kidney, heart, gonad, and [20,41]. The samples were analysed in triplicate for each

Table 3 Sampling sites of the environmental matrices (soil, nettle leaves, snail body) Sampling site Geographical coordinates Location/county (latitude/longitude) THM1 Timisoara city Landfill 45.6765° lat. N; 21.1626° long. E (Parta-Sag/Timis county) THM2 South Industrial Platform 45.7112° lat. N; 21.1968° long. E (Timisoara/Timis county) THM3 North Industrial Platform 45.7420° lat. N; 21.1886° long. E (Timisoara/Timis county) THM4 East Industrial Platform 45.7763° lat. N; 21.2517° long. E (Timisoara/Timis county) THM5 Timisoara, Communal Road 64 (DC64) 45.7787° lat. N; 21.2762° long. E (Timisoara/Timis county) THM6 National Road Resita-Caransebes (DN58) 45.3496° lat. N; 21.9196° long. E (Caras-Severin county) THM7 Principal Street 45.5053 lat. N; 22.3371 long. E (Otelu Rosu/Caras-Severin county) THR Salbagelu Nou 45.5667° lat. N ; 22.0812° long. E (Caras-Severin county) Nica et al. Chemistry Central Journal 2012, 6:55 Page 12 of 15 http://journal.chemistrycentral.com/content/6/1/55

location, and 20 snails were used for each batch. The were collected (25 g/sample in triplicate) from the top empty shells were dried with sterile paper towels and 15 cm layer after removal of vegetation (grass). The kept for further analysis. fresh soil samples were hand sorted to remove roots and Firstly we aimed to find which of the shell main con- litter and, then dried at room temperature (t = 22°C) for chological features, shell height (SH) or shell weight 7 days. Finally, the samples were disaggregated and (SW), allows a more accurate estimation of shell volume. homogenized before being sieved to 2 mm (soil metal For each population, ten shells were randomly selected concentration analysis), and then stored at ambient to be measured for determining shell height (SH), shell temperature (t = 22°C) for further analysis. weight (SW), and shell volume (SV). SH and SW were compiled from the malacological literature [56]; the Chemical analysis measurements were performed with a digital caliper (YT Chemical analyses were performed separately on each 7201, Yato Electronics Co. Ltd, Guangzhou, China) to trophic compartment (soil, nettle leaves, snail). The snail the nearest 0.01 mm. SV was estimated directly by using meat samples (foot, hepatopancreas) were defrosted a calibrated pipette (vol. = 25 ml, Diffco Din, Witeg, (about 5 g/each sample), and oven dried at 105°C for 24 Germany) to fill the shell with distilled deionized water. hours. Then, the samples were digested in the Muffle Then, we statiscally investigated the presumptive effect furnace (Nabertherm B150, Lilienthal, Germany); the of long-term HM exposure on shell height (SH), relative temperature was stepwise increased up to 550°C till the shell height (RSH), and whorl number (WN). The refer- white ash was formed. The ash was dissolved in 20 mL ence population (R snails) was used as a benchmark to re- of 0.5 N HNO3 and filtered through ash-free filter paper veal intraspecific variation in shell morphology depending before analysis. Finally, each sample volume was brought on the level of Cu, Zn, Cd, and Pb accumulation in the en- to 50 mL with 30 mL HNO3 0.5 N. The dried nettle vironmental matrices (soil, nettle leaves, snail foot, snail samples were similarly processed. hepatopancreas). All the shells were measured to deter- For soil analysis, about 5 g of dried and ground soil mine SH. For each site, the most homogenous 20 shells per each sample was put in polyethylene tubes (Thermo were selected based on the SH value, and measured to de- Scientific Nunc, 30x115 mm, 50 mL). Metals were termine SW and WN. These features were also compiled passed out from soil to solution by wet extraction/pro- from malacological literature [56]. RSH was calculated as ceeding; therefore, each sample was treated with mineral ratio between SH and SW. acids (HNO3 0.5 N) at 1:10 soil/nitric acid solution ratio All the measurements were performed by the same re- for 24 hours. Then, the samples were centrifugated at searcher in the same conditions for all the investigated 1,500 rpm for 15 min., and for each sample 20 mL of sites. Every measurement was repeated five times and supernatant was transferred in a sterile polyethylene only the mean value was taken into account. tube (Thermo Scientific Nunc, 30x115 mm, 50 ml) by using a calibrated pipette (vol. = 25 mL, Diffco Din, Sampling and preparation of soil and vegetation Witeg, Germany). Finally, each sample volume was bring At each sampling point numerous Roman snails were up to 50 mL with HNO3 0.5 N. observed feeding on nettle (Urtica dioica). In addition, Measurements of HM (Cu, Zn, Cd, Pb) concentrations irregular holes with smooth edges were often found on in environmental matrices (soil, nettle leaves, snail foot, nettle leaves in sampling areas revealing H. pomatia pre- snail hepatopancreas) were carried out in the Environ- ference for this perennial plant species; therefore, the mental Research Test Laboratory, Banat`s University of selected food chain included nettle as the main food Agricultural Sciences and Veterinary Medicine from source of Roman snail (Helix pomatia). For each loca- Timisoara, Romania. All samples were weighed on an tion three samples from the top leaves (25 g each) were analytical balance (model TP-214, Denver Instrument collected, separated, and rinsed in distilled water to wash Gmbh, Göttingen, Germany) to the nearest 0.1 mg. The off potential air pollutants. The nettle leaves were oven digestion solutions (HNO3 0.5 N) were prepared from dried at 105°C to constant weight. The dried samples Merck`Suprapur` nitric acid (65%, ρ = 1.39 g/cm3, Merck were crushed with a mortar (Isolab SL-1372), passed KGaA, Darmstadt, Germany). The metal concentrations through a 2 mm sieve, and kept for further analysis in in the filtrate were determined by flame atomic absorp- self-sealing sterile paper pouches at room temperature tion spectrophotometry with high resolution continuum (t = 22°C). source (Model ContrAA 300, Analytik Jena, Germany), Terrestrial gastropods regularly eat soil [40], and fitted with a specific conditions of particular metal, and therefore, they accumulate heavy metals not only via they were expressed as miligram per kilogram dry weight food from the food chain, but also via contaminated soil. (mg kg-1 d.w.). Mix standard solutions (1000 mg/L) of Because Helix pomatia spends its entire life on or in the Cu, Zn, Cd, and Pb - ICP Multielement Standard solu- upper soil horizons, for each location the soil samples tion IV CertiPUR, were purchased from Merck (Merck Nica et al. Chemistry Central Journal 2012, 6:55 Page 13 of 15 http://journal.chemistrycentral.com/content/6/1/55

KGaA, Darmstadt, Germany). Solutions of varying con- Wilcoxon test, df = 1,19). A Brown-Forsythe test (df = centrations were prepared for all metals by diluting sui- 7,153) was conducted to determine if normalized table volumes of standard solutions. Double distilled values meet the assumption of homoscedasticity. One water (spectroscopic pure) was used for the preparation Way Analysis of Variance (Anova, df = 7,153) was car- of reagents and standards. All chemicals were trace ried out to test for significant differences in shell size metal grade (Suprapur). All glassware was treated with and whorl number among different snail populations. Pierce solution 20% (v/v), rinsed with cold tap water fol- Post hoc analysis used Dunnett’s test (df =1,39). This lowed by 20% (v/v) nitric acid and then rinsed with test is used when several treatment means are each double-distilled water. For quality control purposes all compared to a control/reference mean. Because the blanks and duplicate samples were analyzed during the sample sizes were different (SH, RSH, WN: n = 20; procedure. NCS Certified Reference Material-DC 85104a HM accumulation: n = 3), for each sample only the and 85105a (China National Analysis Center for Iron&- mean value of each parameter was taken into account Steel) was analyzed for quality assurance. The percent to conduct a bivariate analysis (Pearson correlation recovery means were: Cu (102%), Zn (103%), Cd (104%), matrix, 3x16 matrix, df = 14). Pb (96%). The variation coefficients were below 10%. All Statistical analyses were performed by using Statistica detection limits (mg kg-1) were assessed by using the 10 [57] and Past [58] software packages. All data are pre- calibration curve method: Cu (0.12), Zn (0.21), Cd sented as the mean ± SD. A p value < 0.05 was consi- (0.02), Pb (0.04). The blank reagent and standard refer- dered significant. ence soil were included into each sample batch to verify Abbreviations the accuracy and precision of the digestion procedure HM: Heavy metals; THR: Reference sampling site; THM1 – THM7: Sampling and also for subsequent analyses. sites 1-7; SH: Shell height; SW: Shell width; SV: Shell volume; RSH: Relative shell height; WN: Shell whorl number; NC: Normal content; ATV: The alert threshold level. Statistical analysis Because of the small size of samples (n = 3), for each en- Competing interests vironmental matrix (soil, nettle leaves, snail foot and The authors declare that they have no competing interests. hepatopancreas), a non-parametric test (Kruskal-Wallis Authors' contributions one-way ANOVA, df = 7,17) was used for assessing dif- DVN, MB, IG, MH, and DMB*, have contributed mainly to the study design, ferences in HM accumulation among investigated loca- collection of data, sampling of soil, vegetation, and snails, chemical analyses, interpretation of results and preparation of paper. All authors read and tions. Similarly, it was determined whether HM approved the final manuscript. accumulation in snail body varied between the snail foot and hepatopancreas (Kruskal-Wallis one-way ANOVA, Acknowledgments All authors have equal rights and have contributed evenly to this paper. The df = 7,17). To test for significant relationships between present work was funded by the project “Post- doctoral School Of HM concentrations within a food chain compartment a Agriculture And Veterinary Medicine Posdru/89/1.5/S/62371”, co-financed by bivariate analysis was carried out (Pearson correlation the European Social Fund through the Sectorial Operational Programme for the Human Resources Development 2007–2013. We are grateful for the matrix, 16x16 matrix, df = 14). For each environmental anomymous reviewers’ constructive comments and recommendations, matrix (n = 24), HM accumulation was checked to see if which increased the quality of the present article. In addition, the first author it meets the normality assumptions (Shapiro-Wilcoxon (DVN) wish to thank D.C. for continuous support throughout his scientific career. test, df = 1,23). The data were then normalized by decimal logarithmation (Shapiro-Wilcoxon test, df = 1,23). Author details 1 Finally, we performed a cluster analysis to classify the Banat's University of Agricultural Sciences and Veterinary Medicine from Timisoara, Faculty of Animal Sciences and Biotechnologies, TimisoaraCalea investigated sites depending on the level of HM accumu- Aradului 119RO 300645, Romania. 2Banat's University of Agricultural Sciences lation in snail hepatopancreas. Such statistical analysis and Veterinary Medicine from Timisoara, Faculty of Food Processing 3 produces a 'dendrogram' showing how data points Technology, Calea Aradului 119, RO 300645, Timisoara, Romania. Banat's University of Agricultural Sciences and Veterinary Medicine from Timisoara, (rows) can be clustered. We used the constrained Ward's Faculty of Agriculture, Timisoara, RO 300645Calea Aradului 119Romania. method (df = 95) because this alghoritm allows the clus- ters to be joined such that increase in within-group var- Received: 29 March 2012 Accepted: 31 May 2012 Published: 15 June 2012 iance is minimized. To estimate SV from linear shell dimensions (SH, References SW), measured values (n = 80) were normalized by 1. Lovett GM, Burns DA, Driscoll CT, Jenkins JC, Mitchell MJ, Rustad L, Shanley JE, Likens GE, Haeuber R: Who needs environmental monitoring? Front decimal logarithmation (Shapiro-Wilcoxon test, df = Ecol Environ 2007, 5:253–260. 1,79). The relationships existing among them were 2. Wharfe J: Hazardous chemicals in complex mixtures – a role for direct assessed by a simple correlation analysis (Pearson toxicity assessment. Ecotoxicology 2004, 13:81–88. 3. Murariu M, Dragan ES, Drochioiu G: Electrospray ionization mass matrix, df = 8). Next, SH, RSH, and WN values (n = 20) spectrometric approach of conformationally-induced metal binding to were normalized by decimal logarithmation (Shapiro- oligopeptides. Eur J Mass Spectrom 2010, 16:511–521. Nica et al. Chemistry Central Journal 2012, 6:55 Page 14 of 15 http://journal.chemistrycentral.com/content/6/1/55

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doi:10.1186/1752-153X-6-55 Cite this article as: Nica et al.: Bioaccumulative and conchological assessment of heavy metal transfer in a soil-plant-snail food chain. Chemistry Central Journal 2012 6:55.

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